Article Figures & Data

Figures

Theoretical dependence of FRET on acceptor concentration for different fluorophore distributions. a, If all donor and acceptor fluorophores are clustered, FRET is independent of acceptor concentration. b, If all fluorophores are randomly distributed, FRET increases with increasing acceptor concentration and is independent of the ratio of donor:acceptor. c, If a fluorophore population is a mixture of clustered and randomly distributed molecules, FRET increases with increasing acceptor concentration, but, for a given acceptor surface density, it also increases with higher ratios of acceptor:donor fluorophores.

Tagging with YFP or CFP does not significantly interfere with the intracellular processing or with the interactions of the class I molecules with the TAP complex. Cells expressing A2-YFP (a), untagged HLA-A2 (b), A2-T134K-CFP (c), or untagged A2-T134K (d) were metabolically labeled with a mixture of [35S]methionine and cysteine and chased in nonradioactive medium for the indicated intervals. The cells were lysed in 0.5% Triton X-100 and immunoprecipitated with mAb BB7.2. The samples were digested with Endo H. Acquisition of resistance to Endo H indicates that the sugar moieties of the labeled molecules have been processed by enzymes residing in the medial Golgi. R and S refer to the Endo H-resistant and the Endo H-sensitive forms of the proteins, respectively. e, Cells expressing A2-YFP or A2-T134K-CFP were metabolically labeled as before and lysed in 1% CHAPS. They were immunoprecipitated with either anti-TAP1 antiserum or BB7.2 mAb and treated with Endo H. R and S refer to the Endo H-resistant and the Endo H-sensitive forms of the proteins, respectively. f, Cells expressing A2-YFP were treated with 100 μM lactacystin or 250 μM Tax peptide for 1.5 h, metabolically labeled, and lysed in 1% CHAPS. Equal amount of lysates was immunoprecipitated with anti-TAP1 antiserum or BB7.2 mAb (as a positive control). Untransfected cells were used as a negative control. Mix refers to the immunoprecipitate from a 1:1 lysate mixture of radiolabeled untreated cell and nonradioactive cells treated with 250 μM Tax peptide for 1.5 h as a control for peptide-induced A2-YFP dissociation from TAP in vitro.

FRET was measured by imaging the increase in CFP fluorescence after photobleaching YFP. Images are pseudocolored for fluorescence intensity, and the color scheme is shown in the bar on the lower right. a, Donor fluorescence before bleaching the acceptor (CFPpre); b, acceptor fluorescence before photobleaching (YFPpre); c, donor fluorescence after bleaching the acceptor (CFPpost); and d, acceptor fluorescence after the bleach (YFPpost).

Dependence of FRET on acceptor (YFP) concentration in cells expressing CFP and YFP physically linked to the cytoplasmic tail of HLA-A2. Each point represents the calculated FRET and the mean value of YFP fluorescence, from a 5 × 5-pixel square placed on the nuclear membrane of the cells. In untreated cells, FRET was independent of YFP concentration (□). This distribution did not change upon treatment with 100 μM lactacystin for 1.5 h () or 250 μM Tax peptide for 1.5 h (•).

Dependence of FRET on YFP concentration for different acceptor:donor ratios: YFP:CFP, ∼1:2 (□); YFP:CFP, ∼1:1 (); YFP:CFP, ∼2:1 (•). a, In cells expressing A2-YFP and A2-CFP, FRETdepended both on YFP concentration and the ratio of YFP:CFP. b, In cells expressing A2-T134K-YFP and A2-T134K-CFP, FRET depended both on YFP concentration and the ratio of YFP:CFP. c, In cells expressing A2-YFP and A2-T134K-CFP, FRET depended on YFP concentration, but was independent of the ratio of YFP:CFP. d, The mean FRET (%) for YFP concentration in the range 1500–2500 fluorescence units for YFP:CFP ratios 1:1 and 2:1, as well as the 98% confidence limits (bars), were plotted for the mixtures of A2-YFP and A2-CFP (▨), A2-T134K-YFP and A2-T134K-CFP (▧), and A2-YFP and A2-T134K-CFP (▩).

Dependence of FRET on YFP concentration after treatment with 100 μM lactacystin for 1.5 h for different acceptor:donor ratios: YFP:CFP, ∼1:2 (□); YFP:CFP, ∼1:1 (); YFP:CFP, ∼2:1 (•). a, In cells expressing A2-YFP and A2-CFP, FRET depended on YFP concentration but was independent of the YFP:CFP ratio. b, In cells expressing A2-T134K-YFP and A2-T134K-CFP, FRET depended both on YFP concentration and the ratio of YFP:CFP. c, In cells expressing A2-YFP and A2-T134K-CFP, FRET depended on YFP concentration, but was independent of the YFP:CFP ratio. d, The mean FRET (%) for YFP concentration in the range 1500–2500 fluorescence units for YFP:CFP ratios 1:1 and 2:1, as well as the 98% confidence limits (bars), were plotted for the mixtures of A2-YFP and A2-CFP (▨), A2-T134K-YFP and A2-T134K-CFP (▧), and A2-YFP and A2-T134K-CFP (▩).

Dependence of FRET on YFP concentration after addition of 250 μM Tax peptide for 1.5 h for different acceptor:donor ratios: YFP:CFP, ∼1:2 (□); YFP:CFP, ∼1:1 (); YFP:CFP, ∼2:1 (•). In cells expressing A2-YFP and A2-CFP (a), A2-T134K-YFP and A2-T134K-CFP (b), or A2-YFP and A2-T134K-CFP (c), FRET depended both on YFP concentration and the ratio of YFP:CFP. d, The mean FRET (%) for YFP concentration in the range 1500–2500 fluorescence units for YFP:CFP ratios 1:1 and 2:1, as well as the 98% confidence limits (bars), were plotted for the mixtures of A2-YFP and A2-CFP (▨), A2-T134K-YFP and A2-T134K-CFP (▧), and A2-YFP and A2-T134K-CFP (▩).